Alcohol dehydrogenase (ADH) isoenzymes, as well as ADH from different sources vary greatly in their substrate specificity with respect to steroids. Activity with 3 beta-hydroxysteroids is confined to certain isoenzymes in some species (ADH SS in the horse) in others (man) all isoenzymes are active. Properties of the isoenzymes with regard to steroid activity may throw some light on the physiological role of ADH. We are investigating the mode of binding of steroids and clssical ADH substrates to the catalytic site of rat liver ADH by employing steroidal and non-steroidal inhibitors as experimental probes. Properties of rat liver ADH are investigated and compared with those of the horse liver ADH SS. While the structure-function relationship of only three of the nine electrophoretically separable bands of horse liver ADH is known, neither the origin nor function of the five remaining components is understood. Our current efforts are concerned with the separation and characterization of these components. Rat liver ADH is unique in that there are no isoenzymes; it is structurally homogeneous and has high activity with 3 beta- hydroxysteroids. Since rat is an experimental animal used most frequently in studies on effects and metabolism of ethanol we have undertaken investigation of kinetics of rat liver ADH to establish the reaction mechanism with ethanol-acetaldehyde and a pair of steroid substrates. A method has been developed for semiquantitative estimation of the human ADH isoenzymes in liver biopsy samples. Concurrently with the projects described above the method will be used to examine the electrophoretic separation patterns of ADH from alcoholics and non- alcoholics.